1. Field of the Invention
The present invention relates to amplification and detection of a specific sequence contained in target nucleic acid, DNA, detection of polymorphism in a gene, analysis of single nucleotide polymorphism, etc.
2. Background Art
As a method for analyzing polymorphism in a gene, there are the Invader method (Lyamichev, et al., Science 260, 778-783 (1993)) and a single strand conformation polymorphism analysis (SSCP analysis).
In the Invader method, nucleic acid fragments having base sequences specific to two different allelic genes are labeled with different types of fluorescent substances. The nucleic acid fragments, when they completely agree with the target allelic genes, can be detected by detecting the fluorescent substance. Non-patent Document 1 has succeeded in miniaturizing a reaction chamber for the Invader method and reducing the amount of genomic DNA used in the reaction to 0.2 ng (about 40 copies).
In the SSCP method, a two-stranded nucleic acid fragment is amplified, split into single strands, denatured and then subjected to electrophoresis. In this manner, the difference of a single base can be distinguished as a conformational difference and thus analyzed. Furthermore, in the SSCP method, chromosomal deletion, in other words, loss of heterozygosity (LOH) can be detected by determining a quantitative ratio of two different allelic genes containing polymorphism loci, comparing a test sample with a standard sample for the quantitative ratio of the two different allelic genes.
Furthermore, a minute amount of tumor cells is sampled by the single cell sampling system (CLONIS/OL; manufactured by Olympus Corporation) or Laser Capture Microdissection (LCM) Systems (manufactured by BM Equipment Co., Ltd.) and a minute amount of a gene (nucleic acid) is extracted from the tumor cells, amplified and analyzed.
In the prior Art of Non-patent Document 2, it is shown that in sampling nucleic acid for analyzing a microsatellite polymorphism of an allelic gene, the lower the number of copies of the nucleic acid in an aliquot, the larger the degree of variation (dispersion) from a statistical point of view, with the result that the reproducibility of an analysis for detecting an increase or decrease of a chromosome is not high. To overcome this problem, time-release PCR has been proposed in the prior art. In the time-release PCR, a nucleic acid, even if it is present in a low number of copies, can be amplified. Therefore, analysis can be directly performed without performing a step of sampling or dispensing a minute amount of nucleic acid.
[Non-patent Document 1] Abstract of the 26th annual meeting of the Japanese Biology Society of Japan, p778, 2PC-104;
[Non-patent Document 2] Slebos et al., Laboratory Investigation (2004) 84, 649-657.